The broad, long-term objectives of this revised proposal are to characterize a novel aspect of the regulation of MAP kinase-dependent signal transduction by dual specificity phosphatases (DSPs). The JNKs are a subgroup of the MAP kinases that are activated in response to pro-inflammatory cytokines and environmental stresses and are implicated in the regulation of proliferation and apoptosis. They are recognized as therapeutic targets for treatment of several major human diseases including cancer, inflammation, as well as neurodegenerative and metabolic diseases. In contrast to studies to date that have focused on the downregulation of JNK signaling by DSPs, extensive preliminary data are presented showing that 2 DSPs, JSP1 (Jnk Stimulatory Phosphatase 1) and the closely related enzyme DSP18/JSP2, have the potential to augment the activation of JNK. The objective of the proposed studies is to define the role of these DSPs in regulation of the JNK pathway, testing the hypothesis that they may function as determinants of specificity in JNK signaling responses. The Specific Aims are: (1) To analyze the effects on cell signaling of altering expression of JSP1 in cell models, including the use of RNA interference. (2) To conduct a structure-function analysis of JSP1, focusing on the identification of its physiological substrates. (3) To characterize DSP18, the closest relative of JSP1, to determine whether it also functions as a regulator of JNK signaling. (4) To characterize the function of JSP1 through analysis of the phenotype of JSP1 knockout mice and analysis of JNK signaling in tissues and cells derived from these mice. The health relatedness of this research lies in the potential therapeutic implications. The JNKs are recognized as therapeutic targets for several major human diseases. The large number of JNK isoforms, together with their importance in a wide variety of cell functions, suggests that drugs designed to inhibit the JNKs directly at the active site may exert broad-ranging effects thereby limiting their utility. In contrast, if JSP1 or DSP18/JSP2 are shown to regulate the activation of specific JNK isoforms or to activate JNK in response to specific stimuli, an inhibitor of these DSPs may attenuate JNK signaling in a more restricted context, possibly enhancing its therapeutic potential.